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J. Biol. Chem., Vol. 280, Issue 2, 1016-1023, January 14, 2005

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Insulin-induced -Arrestin1 Ser-412 Phosphorylation Is a Mechanism for Desensitization of ERK Activation by G_i-coupled Receptors^*

Christopher J. Hupfeld, Jamie L. Resnik, Satoshi Ugi, and Jerrold M. Olefsky¶||**

From the Department of Medicine, Division of Endocrinology and Metabolism and the Department of Reproductive Medicine, University of California, San Diego, La Jolla, California 92093, the ^¶Veterans Affairs Hospital, Research Service, San Diego, California 92161, and ^||The Whittier Diabetes Institute, La Jolla, California 92037

Received for publication, April 2, 2004 , and in revised form, October 12, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
-Arrestin1 is an adapter/scaffold for many G protein-coupled receptors during mitogen-activated protein kinase signaling. Phosphorylation of -arrestin1 at position Ser-412 is a regulator of -arrestin1 function, and in the present study, we showed that insulin led to a time- and dose-dependent increase in -arrestin1 Ser-412 phosphorylation, which blocked isoproterenol- and lysophosphatidic acid-induced Ser-412 dephosphorylation and impaired ERK signaling by these G protein-coupled receptor ligands. Insulin treatment also led to accumulation of Ser-412-phosphorylated -arrestin1 at the insulin-like growth factor 1 receptor and prevented insulin-like growth factor 1/Src association. Insulin-induced Ser-412 phosphorylation was partially dependent on ERK as treatment with the MEK inhibitor PD98059 inhibited the insulin effect (62% reduction, p = 0.03). Inhibition of phosphatidylinositol 3-kinase by wortmannin did not have a significant effect (9% reduction, p = 0.41). We also found that the protein phosphatase 2A (PP2A) was in a molecular complex with -arrestin1 and that the PP2A inhibitor okadaic acid increased Ser-412 phosphorylation. Concomitant addition of insulin and okadaic acid did not produce an additive effect on Ser-412 phosphorylation, suggesting a common mechanism. Small t antigen specifically inhibited PP2A, and in HIRcB cells expressing small t antigen, -arrestin1 Ser-412 phosphorylation was increased, and insulin had no further effect. Insulin treatment caused increased -arrestin1 Ser-412 phosphorylation, which blocked mitogen-activated protein kinase signaling and internalization by -arrestin1-dependent receptors with no effect on -adrenergic receptor G_s-mediated cAMP production. These findings provide a new mechanism for insulin-induced desensitization of ERK activation by G_i-coupled receptors.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Many hormone signaling systems are mediated by G protein-coupled receptors (GPCRs),¹ and several have been found to utilize -arrestin as a key regulatory protein (1–8). In addition, the insulin-like growth factor-1 (IGF-1) receptor, a receptor tyrosine kinase, has recently been found to activate MAP kinase signaling in a G_i- and -arrestin1-dependent manner (9). Thus, changes in -arrestin function can have widespread implications for hormone signaling. We have recently found that insulin treatment is associated with down-regulation of -arrestin1 protein (10) as well as phosphorylation of -arrestin1 Ser-412 (11), representing possible new mechanisms of insulin-induced impairment of -arrestin1-dependent GPCR signaling.

Activation of adenylate cyclase and generation of cAMP following ligand binding to some GPCRs coupled to G_s is attenuated once -arrestin1 binds to the activated GPCR and sterically uncouples the receptor from further interaction with G_s (5). Other GPCRs, however, utilize -arrestin as a scaffold for assembly of a multiprotein signaling complex that leads to activation of ERK1/2 (12–14). In addition, -arrestin interacts with clathrin, targeting activated receptors to clathrin-coated pits for endocytosis (6, 15, 16). Therefore, -arrestin performs a dual role during intracellular signaling by GPCRs: desensitizing signals leading to adenylate cyclase activation while coordinating and enhancing signals leading to ERK1/2 activation.

As one would predict from the known functions of -arrestin discussed above, a reduction of -arrestin protein content using antisense oligonucleotide (17), knock-out gene technology (18), small interfering RNA (19) or by insulin treatment (11) is associated with both impaired desensitization of G_s signaling (G_s signal "supersensitization") and impaired ERK activation.

-Arrestin1 function during G protein-mediated ERK signaling is also regulated by phosphorylation of the carboxyl terminus Ser-412 residue. Dephosphorylation of Ser-412 is required for the isoproterenol-stimulated association between -arrestin1 and Src, a non-receptor tyrosine kinase involved in 1- and 2-adrenergic receptor (AR) mitogenic signaling (12). In addition, Ser-412 dephosphorylation is also required for -arrestin1/clathrin association, which is essential for 2AR internalization (16). Mutants of -arrestin1 that have a serine to aspartic acid substitution (S412D) and thus mimic the phosphorylated state at the 412 residue have reduced affinity for Src and for clathrin and function as dominant negative inhibitors of G protein-mediated MAP kinase signaling (16). The mechanism behind the regulation of -arrestin1 Ser-412 phosphorylation/dephosphorylation has not been described. We have recently shown that insulin treatment leads to -arrestin1 degradation (10) as well as increased -arrestin1 Ser-412 phosphorylation (11). In these studies, insulin-induced changes in -arrestin1 were associated with increased AR/G_s signaling (due to loss of G_s signal desensitization) and with impaired G_i-mediated MAP kinase activation (due to defective -arrestin/Src and -arrestin/clathrin interaction). However, the relative importance of insulin-induced protein degradation versus insulin-induced Ser-412 phosphorylation in these processes remains unclear.

In addition, the mechanism of regulation of Ser-412 phosphorylation is unknown. Previous studies have described a role for ERK during Ser-412 phosphorylation (16, 20), while others have found the protein phosphatase 2A (PP2A) and PP2B in complexes that contain -arrestin (21). In this regard, we have recently found that insulin-induced reduction of PP2A activity is involved in the desensitization of ERK signaling (22), but the role of PP2A in regulating -arrestin1 Ser-412 phosphorylation has not been described.

In the current study, we present a new mechanism of insulin-induced desensitization of GPCR signaling. Thus, insulin-induces -arrestin1 Ser-412 phosphorylation, and this disrupts many of the known -arrestin1 functions in GPCR signaling. In addition, we demonstrate a novel role for PP2A in this process.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—IGF-1, lysophosphatidic acid (LPA), PD98059, wortmannin, and okadaic acid were purchased from Calbiochem. CGP-12177 was purchased from Pierce. Anti--arrestin1 was from Transduction Laboratories. Anti-phospho--arrestin1 (Ser-412) and anti-phospho-Akt (Ser-473) were from Cell Signaling Technology. Anti-phospho-ERK1/2, anti-PP2, and anti-Src were from Santa Cruz Biotechnology. Insulin, isoproterenol, BRL 37344, and all other chemicals were from Sigma.

Cell Culture—Confluent 3T3-L1 preadipocytes were differentiated and maintained at 37 °C with 10% CO₂ in Dulbecco's modified Eagle's medium supplemented with 4.5g/liter glucose, 10% fetal calf serum, 0.1% Glutamax, and penicillin/streptomycin. On day 1, medium was supplemented with 3-isobutyl-1-methylxanthine (0.11 mg/ml), dexamethasone (0.2 µg/ml), and insulin (5 ng/ml). On day 4, the differentiation mixture was replaced with regular medium. On day 7, cells were reseeded, and experiments were performed on days 10–14 postdifferentiation. Cells were placed in serum-free medium (Dulbecco's modified Eagle's medium with 1.0 g/liter glucose, 0.5% bovine serum albumin, 0.1% Glutamax, and penicillin/streptomycin) for 16 h prior to ligand stimulation unless otherwise indicated.

Immunoprecipitation and Western Blotting—Cells were lysed, and protein concentrations were determined by the Bradford assay as described previously (9). For Western blotting, 25 µg of cellular protein were dissolved in Laemmli buffer supplemented with dithiothreitol and boiled for 5 min. For immunoprecipitations, 500 µg of total protein were diluted to 1 µg/µl in PBS to which 3 µg of the appropriate antibody were added overnight at 4 °C. Immune complexes were captured by adding 40 µl of a 50% protein A/G bead mixture for an additional 1 h. Beads were then washed three times with PBS, dissolved in Laemmli buffer plus dithiothreitol, and boiled for 5 min. Proteins were separated by 7.5–10% SDS-PAGE and then transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% nonfat dry milk in Tris-buffered saline/Tween 20 and probed with the appropriate primary and secondary antibodies. Horseradish peroxidase-conjugated secondary antibody binding was detected by chemiluminescence.

Generation of Small t Antigen-expressing Cell Lines—We have previously described the generation of stable cell lines expressing small t antigen in our laboratory (22). Rat-1 fibroblasts overexpressing human insulin receptors (HIRcB cells) were maintained as described previously (23). A plasmid encoding small t antigen, pCMV5-small t (24), was a gift from Marc C. Mumby (University of Texas Southwestern Medical Center, Dallas, TX). cDNA from pCMV5-small t was digested with HindIII and BamHI and subcloned into pcDNA3.1/Hygro (Invitrogen), which contains the hygromycin B phosphotransferase gene. The resultant plasmid, pcDNA3.1/Hygro-small t, was transfected into HIRcB cells by using Superfect according to the manufacturer's instructions. Stable cell lines were selected in 400 µg of hygromycin B/ml. Clonal cell lines were isolated by limiting dilution and then screened by immunoblotting with anti-small t antibody. Parental cells transfected with pcDNA3.1/Hygro alone (Hyg cells) were used as a control.

Intracellular cAMP Measurement—Intracellular cAMP levels were determined using the cAMP enzyme immunoassay system from Amersham Biosciences as described previously (11) according to manufacturer's instructions.

Ligand Binding Assay—AR internalization was determined by ligand binding assay using CGP-12177, a hydrophilic AR ligand, in the manner described previously (11, 25). In 12-well plates, 3T3-L1 adipocytes were serum-starved overnight in the presence or absence of insulin (1 ng/ml), washed with PBS at 37 °C, and acutely stimulated with isoproterenol (10 µM for 15 min) in starvation medium. The reaction was terminated with rapid washing with ice-cold PBS, and 1µl/ml [³H]CGP-12177 was added to each well in a binding buffer (Dulbecco's modified Eagle's medium, 20 µM HEPES, 10 nM unlabeled CGP-12177, at pH 7.4) and incubated for 4 h at 4 °C. Following washing with PBS, plasma membrane proteins were solubilized via two 10-min incubations in acidic buffer, and the resulting supernatant was assayed for ³H content by scintillation counting. NaOH cell lysis following acid extraction confirmed the retention of CGP-12177 at the cell surface as significant ³H was not detected in cell lysates. Nonspecific binding was determined by competitive inhibition of CGP-12177 binding with a saturating dose of propanolol (20 µM).

Data Quantitation and Statistical Analyses—Densitometry results were quantitated using Kodak Image Station 440 software, and values are expressed as means ± S.E. Data were analyzed using the Student's t test, and p values were assigned with p 0.05 considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Insulin Treatment Leads to a Time- and Dose-dependent Increase of -Arrestin1 Ser-412 Phosphorylation—We have previously shown that insulin treatment (100 ng/ml) for 8–12 h leads to proteasomally mediated degradation of -arrestin1, reducing cellular -arrestin1 function (10). Phosphorylation of Ser-412 can also impair -arrestin1 activity, providing an alternate mechanism to functionally down-regulate this protein.

As shown in Fig. 1, insulin treatment of 3T3-L1 adipocytes led to a time- (Fig. 1, A and B) and dose-dependent (Fig. 1, C and D) increase in Ser-412 phosphorylation of -arrestin1. As indicated in Fig. 1B, the 6-h insulin pretreatment did not cause substantial -arrestin1 protein degradation (reduced 10–15%). In contrast, treatment with isoproterenol and LPA, whose cognate GPCRs require -arrestin1 Ser-412 dephosphorylation for ERK signaling (10), led to Ser-412 dephosphorylation (Fig. 1E). EGF receptor signaling is independent of -arrestin, and EGF stimulation did not have a significant effect on Ser-412 phosphorylation (Fig. 1E).

View larger version (28K): [in this window] [in a new window]   FIG. 1. Insulin treatment is associated with increased -arrestin1 Ser-412 phosphorylation. A, serum-starved 3T3-L1 adipocytes were treated with insulin (100 ng/ml) for the indicated time course, and total cell lysates were subjected to Western blotting as described under "Experimental Procedures." B, results from A were quantified by densitometry as described under "Experimental Procedures," and data represent mean ± S.E. of eight experiments. C, cells were treated with the indicated doses of insulin for 6 h. D, results from C were quantified by densitometry as above, and data represent the mean ± S.E. of three experiments. E, serum-starved 3T3-L1 adipocytes were treated with isoproterenol (Iso, 10 µM), LPA (10 µM), or EGF (10 ng/ml) for the indicated times, and total cell lysates were subjected to Western blotting. *, p 0.05; **, p 0.01. IB, immunoblot; P-, phospho-.

View larger version (33K): [in this window] [in a new window]   FIG. 2. Insulin treatment inhibits GPCR ligand-mediated Ser-412 dephosphorylation. A and C, 3T3-L1 adipocytes were serum-starved for 6 h in the presence or absence of insulin (Ins, 100 ng/ml). Cells were washed once in 37 °C PBS and then treated with isoproterenol (Iso, 10 µM) or LPA (10 µM) for 15 min where indicated. B, densitometric quantitation for A, as mean ± S.E., from two separate experiments. D, same conditions as A with subsequent treatment with EGF (10 ng/ml) for 5 min. E, same conditions as A with subsequent treatment with the 3AR-specific ligand BRL 37344 (1 µM) for 30 min. IB, immunoblot; P-, phospho-.

Insulin Treatment Increases IGF-1R-associated Ser-412 Phosphorylated -Arrestin1, Prevents IGF-1R/Src Association, and Blocks IGF-1-mediated ERK Activation—-Arrestin1 is a scaffold for several signaling proteins involved in G protein-mediated ERK activation. Best characterized are the interactions between -arrestin1 and Src, a non-receptor tyrosine kinase, and clathrin, a protein involved in receptor internalization. Interaction between -arrestin1 and these proteins requires dephosphorylation at the -arrestin1 Ser-412 site. We have previously shown that IGF-1 treatment leads to -arrestin1 recruitment to the IGF-1R and that IGF-1R-mediated ERK signaling is pertussis toxin-sensitive (dependent on G_i) (9) and -arrestin1-dependent (10). Others have shown that IGF-1R-mediated ERK signaling is Src-dependent (30), and internalization of the IGF-1R is -arrestin- and clathrin-dependent (15).

Our preliminary studies showed that, unlike isoproterenol and LPA, IGF-1 treatment did not lead to detectable Ser-412 dephosphorylation in total cell lysates (not shown). We next determined whether the -arrestin1 recruited to the IGF-1R following IGF-1 treatment is Ser-412-phosphorylated. We measured the amount of Ser-412-phosphorylated -arrestin1 in IGF-1R immunoprecipitates and did not detect significant amounts either before or after IGF-1 treatment (Fig. 3A). In contrast, insulin pretreatment greatly increased the amount of Ser-412-phosphorylated -arrestin1 associated with the IGF-1R following IGF-1 treatment. Strikingly the same insulin treatment protocol blocked IGF-1R/Src association (Fig. 3B) and reduced IGF-1-mediated ERK phosphorylation (Fig. 3C). Thus, IGF-1 normally recruits dephosphorylated -arrestin1 to the receptor, which is fully competent to associate with Src and relay signals to ERK. In contrast, after insulin pretreatment, IGF-1 recruits Ser-412-phosphorylated -arrestin1 to the receptor, which cannot couple to Src, and displays impaired downstream signaling.

View larger version (31K): [in this window] [in a new window]   FIG. 3. Insulin treatment increases IGF-1R associated Ser-412-phosphorylated -arrestin1, prevents IGF-1R/Src association, and blocks IGF-1-mediated ERK activation. A and C, serum-starved 3T3-L1 adipocytes were pretreated with insulin (100 ng/ml) for 6 h. Cells were then washed with PBS and treated with IGF-1 (100 ng/ml) for 5 min. Total cell lysates were immunoblotted as described under "Experimental Procedures." B, following the above treatments, cell lysate proteins were immunoprecipitated with an anti-IGF-1R antibody as described under "Experimental Procedures." IB, immunoblot; P-, phospho-; IP, immunoprecipitation.

View larger version (21K): [in this window] [in a new window]   FIG. 4. Insulin-induced -arrestin1 Ser-412 phosphorylation involves ERK but not phosphatidylinositol 3-kinase. A, serum-starved 3T3-L1 adipocytes were treated with insulin (100 ng/ml), PD98059 (PD, 20 µM), and wortmannin (WM, 100 nM) either alone or in combination for 6 h. Total cell lysates were obtained, and Western blotting was performed as described under "Experimental Procedures." B, data were quantified by densitometry and are displayed as the mean ± S.E. for eight experiments. Data are adjusted for the effect of PD98059 alone (1.1–1.2-fold increase, data not shown). C and D, serum-starved 3T3-L1 adipocytes were treated with PD98059 (B) or wortmannin (C) for 30 min prior to the addition of insulin (100 ng/ml) for the indicated times. Western blotting of total cell lysates was performed as described under "Experimental Procedures," and each blot is representative of four separate experiments. *, p 0.05. IB, immunoblot; P-, phospho-; Ins, insulin.

First we found that -arrestin1 and PP2A were associated in the basal state and that isoproterenol treatment led to an increase in this association in 3T3-L1 adipocytes (Fig. 5A), suggesting that PP2A is involved in isoproterenol-mediated Ser-412 dephosphorylation. If PP2A can decrease Ser-412 phosphorylation, inhibition of PP2A activity should lead to an increase in Ser-412 phosphorylation. Low (1 µM) concentrations of okadaic acid have been shown to specifically inhibit PP2A (33), and when cells were incubated with 1 µM okadaic acid, Ser-412 phosphorylation was clearly increased (Fig. 5B). When insulin and okadaic acid were added together, insulin did not significantly increase Ser-412 phosphorylation above the level induced by okadaic acid alone (Fig. 5, C and E), suggesting either that insulin and okadaic acid act through a common mechanism or that okadaic acid treatment alone leads to stoichiometric phosphorylation at Ser-412, which cannot be enhanced further. In addition, treatment of 3T3-L1 adipocytes with okadaic acid prevented isoproterenol-mediated dephosphorylation of Ser-412 (Fig. 5, D and E), suggesting that isoproterenol treatment requires activation of PP2A to induce Ser-412 dephosphorylation.

View larger version (30K): [in this window] [in a new window]   FIG. 5. PP2A associates with -arrestin1, and treatment with the PP2A inhibitor okadaic acid leads to increased Ser-412 phosphorylation. A, serum-starved 3T3-L1 adipocytes were treated with isoproterenol (Iso, 10 µM) for 0–10 min. Cell lysate proteins (500 µg) were immunoprecipitated overnight with either anti--arrestin1 antibody or with anti-PP2A antibody, and the resulting immune complexes were captured on protein A/G beads. Western blotting was then performed using the indicated antibodies. B, serum-starved 3T3-L1 adipocytes were treated with either okadaic acid (OA, 1 µM) or insulin (Ins, 100 ng/ml) for the indicated times, and Western blotting of total cell lysates was performed as described under "Experimental Procedures." C, serum-starved 3T3-L1 adipocytes were treated with either okadaic acid (OA, 1 µM) alone or with okadaic acid plus insulin (Ins, 100 ng/ml) for 6 h. D, serum-starved 3T3-L1 adipocytes were treated with isoproterenol (Iso, 10 µM) for 30 min. Where indicated, cells were pretreated with okadaic acid (OA, 1 µM) for 6 h prior to the addition of isoproterenol. Total cell lysates were Western blotted as described under "Experimental Procedures." E, densitometry from at least two separate experiments from B–D as mean ± S.E. IB, immunoblot; P-, phospho-; IP, immunoprecipitation.

View larger version (25K): [in this window] [in a new window]   FIG. 6. Insulin-induced Ser-412 phosphorylation is prevented in small t antigen-expressing cells. A, HIRcB cells stably expressing either pcDNA/Hyg alone (Hyg) or pcDNA/Hyg-small t antigen (ST) were serum-starved and then treated with insulin (100 ng/ml) for the indicated times. Total cell lysates were Western blotted as described under "Experimental Procedures." B, densitometry from three experiments described in A. Black bars represent Hyg cells, and white bars represent ST cells. IB, immunoblot; P-, phospho-.

View larger version (19K): [in this window] [in a new window]   FIG. 7. Insulin-induced -arrestin1 Ser-412 phosphorylation impairs -adrenergic receptor internalization. 3T3-L1 adipocytes were serum-starved in the presence or absence of insulin (Ins, 1 ng/ml) for 6 h. A, Western blotting was performed on total cell lysates as described under "Experimental Procedures." B, band densitometry from four separate experiments is shown as mean ± S.E. C, following insulin (1 ng/ml) treatment, cells were washed and then stimulated with isoproterenol (Iso, 10 µM) for the indicated times. Intracellular cAMP levels were determined using an enzyme-linked immunoassay system from Amersham Biosciences according to the manufacturer's instructions. The concentration of cAMP is expressed as fmol of cAMP/µg of protein. The solid line (—) represents isoproterenol alone; the dashed line (- -) represents insulin (1 ng/ml) pretreatment for 6 h followed by isoproterenol treatment. Insulin pretreatment alone had no effect on [cAMP] (data not shown). Experiments were performed in triplicate, and three independent experiments were performed of which the displayed graph is representative. D, AR internalization was determined by a ligand binding assay using the non-internalized AR ligand [3H]CGP-12177 as described under "Experimental Procedures." Each experiment was performed in triplicate, and the displayed graph is representative of three separate experiments. IB, immunoblot; P-, phospho-.

View larger version (17K): [in this window] [in a new window]   FIG. 8. Insulin-induced Ser-412 phosphorylation leads to desensitization of -arrestin-dependent, Gi-mediated MAP kinase signaling. A, following insulin (Ins, 1 ng/ml for 6 h) treatment, cells were washed and immediately treated with isoproterenol (Iso, 10 µM for 15 min), LPA (10 µM for 5 min), IGF-1 (100 ng/ml for 5 min), or EGF (10 ng/ml for 5 min). B, band densitometry for each condition is shown as mean ± S.E. for two separate experiments. C, HIRcB cells stably expressing either empty vector (Hyg) or ST were treated with LPA (10 µM) for the indicated times. Total cell lysates were Western blotted, and densitometry is shown as mean ± S.E. from two separate experiments. IB, immunoblot; P-, phospho-.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

This molecular mechanism was demonstrated using the IGF-1R, which is G_i-coupled and associates with -arrestin1 and Src following IGF-1 stimulation. When IGF-1 stimulation was preceded by insulin pretreatment, the -arrestin1 associated with the IGF-1R was clearly phosphorylated at Ser-412. Perhaps more importantly, insulin pretreatment led to impaired IGF-1-mediated recruitment of Src to the IGF-1R and inhibition of ERK activation by IGF-1. This insulin-induced increase in -arrestin1 Ser-412 phosphorylation represents a novel mechanism for insulin-induced desensitization of G protein-mediated ERK activation.

The importance of -arrestin1 Ser-412 dephosphorylation during initiation of ERK signaling from the activated -adrenergic receptor has been described previously (16). We definitively demonstrated ligand-mediated Ser-412 dephosphorylation in 3T3-L1 adipocytes using two different -arrestin-dependent GPCR ligands, isoproterenol and LPA. In contrast to treatment with GPCR ligands, insulin treatment led to an increase in phosphorylation at Ser-412 and in fact prevented Ser-412 dephosphorylation induced by isoproterenol and LPA. This was associated with desensitization of ERK signaling by both isoproterenol and LPA. Interestingly while stimulation of the IGF-1R, which is G_i-coupled and -arrestin1-dependent for MAP kinase signaling, did not lead to detectable reductions in total cellular Ser-412-phosphorylated -arrestin1, receptor-bound -arrestin1 was clearly dephosphorylated, suggesting that the IGF-1R interacts preferentially with Ser-412-dephosphorylated -arrestin1. Insulin treatment greatly increased the amount of Ser-412-phosphorylated -arrestin1 at the IGF-1R and blocked the association of the IGF-1R and Src. Therefore, ligand-mediated dephosphorylation of Ser-412 plays a critical role in G protein-mediated ERK signaling, and this was prevented by insulin treatment.

-Arrestin1 has an ERK consensus sequence around Ser-412, and a previous study (20) using in vitro phosphorylation and two-dimensional tryptic phosphopeptide mapping identified ERK as the kinase responsible for phosphorylating Ser-412. We found that, using the MEK inhibitor PD98059, insulin-induced Ser-412 phosphorylation in 3T3-L1 adipocytes was reduced by 62%. We conclude that ERK, or another MEK-dependent kinase, was responsible for phosphorylating the Ser-412 site in response to insulin treatment. Interestingly other ERK-activating ligands (isoproterenol, LPA, IGF-1, and EGF) failed to increase Ser-412 phosphorylation, suggesting that a non-ERK mechanism also plays a partial role in determining Ser-412 phosphorylation status.

Our findings suggest that changes in phosphatase activity, specifically PP2A, can have a profound effect on Ser-412 phosphorylation and may partially explain the effects of insulin. PP2A is activated upon isoproterenol stimulation (31), and we found that PP2A co-immunoprecipitated with -arrestin1 in 3T3-L1 adipocytes both at base line and in response to isoproterenol treatment. This is consistent with a previous study in which both PP2A and PP2B were recovered from -arrestin-containing 2AR immunoprecipitates (21), suggesting a role for PP2A in isoproterenol-mediated Ser-412 dephosphorylation. Consistent with this, we found that inhibition of PP2A activity by okadaic acid treatment enhanced Ser-412 phosphorylation and prevented isoproterenol-mediated dephosphorylation of this residue.

We have previously shown that insulin treatment inhibits PP2A activity (22, 32). In the current study, insulin treatment did not lead to further Ser-412 phosphorylation in cells where PP2A was already inactivated by another method. When cells were treated with insulin and okadaic acid together, the effect on Ser-412 phosphorylation was not additive. In addition, we found that insulin treatment had no effect on Ser-412 phosphorylation in HIRcB cells that express the ST, an SV40-derived protein that specifically inhibits PP2A. Interestingly basal phosphorylation of Ser-412 was increased both by okadaic acid treatment and in ST-expressing cells in the absence of ligand stimulation. This suggests that the basal activity of a serine kinase targeting Ser-412 is held in check by PP2A activity and that a balance between the activity of this kinase and PP2A normally regulates Ser-412 phosphorylation levels. It is also possible to conclude from these experiments that okadaic acid treatment and ST expression lead to stoichiometric phosphorylation at Ser-412, and therefore insulin is unable to further increase Ser-412 phosphorylation irrespective of the mechanism involved. However, the results presented here provide a plausible mechanism to explain why ERK activation alone does not fully account for the increase in Ser-412 phosphorylation with insulin treatment.

A previous study examined the stoichiometry of -arrestin1 phosphorylation in human embryonic kidney 293 cells (16). In these cells, -arrestin1 was found to contain 0.84 mol of P_i/mol of protein and that Ser-412 accounts for 90% of -arrestin1 phosphorylation (16, 20). Therefore, in human embryonic kidney 293 cells, Ser-412 phosphorylation stoichiometry is 0.76 mol of P_i/mol of -arrestin1 protein. Our findings in 3T3-L1 adipocytes suggest a similar or slightly lower level of basal Ser-412 phosphorylation as insulin treatment led to a 1.6–1.8-fold increase in phosphorylation at this site. This level of phosphorylation is fully accommodated if the basal stoichiometry in 3T3-L1 adipocytes is 0.6 mol of P_i/mol of -arrestin1 protein.

Treatment with 1 ng/ml insulin, which induced Ser-412 phosphorylation but did not reduce -arrestin1 protein levels, led to impaired MAP kinase signaling by three -arrestin-dependent ligands (isoproterenol, LPA, and IGF-1) and impaired internalization of the AR. Insulin-induced increases in Ser-412 phosphorylation, however, had no effect on AR/G_s-mediated cAMP accumulation and also had no effect on MAP kinase signaling by the -arrestin1-independent EGF receptor. These data are consistent with studies using transfection of -arrestin1 mutants to alter the Ser-412 phosphorylation state. Cells expressing the S412D mutant of -arrestin1, which mimics the phosphorylated state of Ser-412, display impaired -arrestin1 binding to Src and to clathrin, have impaired receptor internalization, and have a defect in ligand-mediated MAP kinase signaling while displaying preserved G_s signal desensitization (12, 16, 21). Thus our findings, in a non-transfected cell system, provide further evidence that desensitization of G_s signaling occurs independently of -arrestin1 Ser-412 phosphorylation and show that Ser-412 phosphorylation following insulin treatment is sufficient to cause desensitization of G protein-mediated ERK signaling.

Possible physiologic consequences of increased Ser-412 phosphorylation would be linked to impaired G protein/ERK signaling as G_s/cAMP/PKA signals are not affected by Ser-412 phosphorylation status. Therefore it is unlikely that adrenergic receptor-mediated inhibition of leptin secretion, which is mediated by a PKA-dependent mechanism, would be affected (36). However, although classically considered a PKA-dependent event, lipolysis may be partially regulated by adrenergic receptor-mediated ERK signaling (37), and therefore -arrestin1 Ser-412 phosphorylation may play a role in this process. Survival and differentiation of preadipocytes and adipocytes may also depend on GPCR-associated, ERK-mediated signaling. Mitogen-induced proliferation of preadipocytes is ERK-dependent, and norepinephrine-induced ERK signaling prevents apoptosis of mature adipocytes (38).

In summary, we found that increased -arrestin1 Ser-412 phosphorylation following insulin treatment was sufficient to disrupt G protein-mediated MAP kinase signaling and was most likely due to the combination of insulin-induced ERK activation and inhibition of PP2A activity. These data represent a new mechanism whereby insulin treatment causes desensitization of G protein-mediated MAP kinase signaling.

	FOOTNOTES

 
^* This work was supported by National Institutes of Health Grant K08 DK65127-01 (to C. J. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence should be addressed: Dept. of Medicine, Division of Endocrinology and Metabolism, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-6073. Tel.: 858-534-6651; Fax: 858-534-6653; E-mail: jolefsky{at}ucsd.edu.

¹ The abbreviations used are: GPCR, G protein-coupled receptor; ERK, extracellular signal-regulated kinase; MAP, mitogen-activated protein; AR, -adrenergic receptor; PP2, protein phosphatase 2; LPA, lysophosphatidic acid; EGF, epidermal growth factor; MEK, MAP kinase/ERK kinase; HIRc, human insulin receptor cDNA; ST, small t antigen; IGF-1, insulin-like growth factor 1; IGF-1R, IGF-1 receptor; PBS, phosphate-buffered saline; PKA, cAMP-dependent protein kinase; SOS, son-of-sevenless.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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